RU2075082C1 - Method of determination of bacterial toxin activity - Google Patents

Abstract

FIELD: medicine, microbiology, toxicology. SUBSTANCE: method involves treatment of luminescent bacteria with exotoxins at different dilutions and their activity is determined by bacteria luminosity inhibition degree after 30 min (not less) of incubation of bacteria with toxin. EFFECT: simplified method, decreased study time. 9 tbl

Description

 The invention relates to the field of microbiology and can be used in the production of bacterial toxins and antitoxins, as well as in research work.

 A known method for determining the activity of exotoxins in a 10-day-old rabbit model (Datta N.K. Habbu M.K. Experimental cholera in Infant Rabbits anethed for chemotherapentic Investigation. -Br. J. Pharmacol, 1955, v. 10, p. 153). The disadvantages of the method are: the inability to determine the amount of titrated toxin, the duration of the response (2 days), the high cost of the study.

 A known method for determining toxin on ligated ileal loops of adult rabbits (Burrous W. Musteiris C. M. Cholera infection and toxin in the rabbit ilal loop. J. Infct. Dis. 1966, v. 116, p. 183). The method is quite informative, but expensive, difficult to execute in 18 hours.

 A known method for determining the activity of the toxin of the syndrome of toxic shock of staphylococci in a rabbit model with intravenous administration (Reeves MW Arko RV Chandler FW et al. Affinity purification of staphylococcal toxic shook syndrome toxin and pathologic effects in rabbits. Infect. Immun. 1986, v. 51, p, . 431 439). The method is expensive, difficult to implement.

 A known method for titration of toxin for intradermal administration to adult rabbits (Croug SP Yamamoto K. Takeda et al. Production of cholera like enterotoxin by vibrio cholera non-01 strain isolated from the environment. - In: Proc. Cholera Res. Symp. Honolulu, Hawaii, 1965 Washington 1965, p. 153). The method is simple to implement, but requires expensive animals, the response time is 18 hours.

 To simplify, reduce the cost of the method and reduce the study time, luminescent bacteria are exposed to exotoxins and the activity of toxins is determined by the degree of inhibition of bacterial luminescence.

 The invention is illustrated by the following examples.

Example 1. In a test tube containing the studied purified concentrated cholera vibrio exotoxin manufactured by the Microbe Saratov Institute series 4, in a quantity of 0.1 ml make a mixture consisting of 0.8 ml of 2% NaCl and 0.1 ml of a solution of luminescent bacteria ( the number of bacteria is provided for by the instruction and is included in the “Ekolyum” kit, produced by the BioChemMack joint venture, designed to determine the content of chemical compounds characteristic of industrial discharges into the environment). The mixture is incubated for 30 min at t 37 ^o C. The control are: cholerogen neutralized by anticholeogenic serum manufactured by the Saratov Institute "Microbe" series 2 with a titer of 3000 AE; cholerogen destroyed by two-hour boiling; as well as saline, in which luminescent bacteria are diluted.

 In parallel, the activity of the toxin and controls is checked on models of an isolated intestinal loop of adult rabbits and in Craig's skin test.

 The contents of the tubes are transferred into cuvettes and the luminosity is recorded on a chemiluminometer PHL-1. The results of the study are presented in table. one.

 Thus, a purified concentrated toxin with an activity of 1600 AE of intestinal units and 5600 AE in Craig's sample inhibits the luminescence of bacteria.

Example 2. In a test tube containing 0.1 ml of partially purified concentrated exotoxin produced by the Saratov Institute "Microbe" series 6, add in an amount of 0.1 ml a mixture consisting of 0.8 ml of 2% NaCl and 0.1 ml of a luminescent solution bacteria. The mixture is incubated for 30 min at t 37 ^o C. The controls are: cholerogen neutralized with anticholeogenic serum produced by the Saratov Institute "Microbe" series 2 with a titer of 3000 AE; cholerogen destroyed by two-hour boiling; as well as physical solution. In parallel, the activity of the toxin and controls is checked on models of an isolated intestinal loop of adult rabbits and in Craig's skin test. Samples are transferred to cuvettes and the intensity of luminescence is recorded on a chemiluminometer PHL-1. The research results are presented in table.2. Table 2 indicates that less active toxins also inhibit the luminescence of bacteria.

Example 3. In a test tube containing crude concentrated exotoxin, in a quantity of 0.1 m make a mixture consisting of 0.8 ml of 2% NaCl and 0.1 ml of luminescent bacteria. The mixture is incubated for 30 min at t - 37 ^o C. The controls are cholerogen neutralized by anticholeogenic serum, manufactured by the Saratov Institute "Microbe" series 2 with a titer of 3000 AE, cholerogen destroyed by two-hour boiling, as well as physical. solution. In parallel, the activity of the toxin is monitored in biological samples. The research results are presented in table.3.

 From the table. Figure 3 shows that crude concentrated cholerogen also inhibits the luminescence of bacteria.

Example 4. In a test tube containing the active supernatant of the toxin-forming strain V. Cholrae 569 B in a quantity of 0.1 ml make a mixture consisting of 0.8 ml of 2% NaCl and 0.1 ml of luminescent bacteria. Active supernatant was obtained by the method of Burrows W. Musteikis GM Cholera infection and toxin in the rabbit ileal loop. J. Infect. Dis. 1966, v. 116, p. 183. The mixture is incubated for 30 min at t 37 ^o C. The controls are active supernatant mixed with anticholeogenic serum produced by the Saratov Institute “Microbe” series 2 with a titer of 3000 AU and the active supernatant boiled for 2 hours. The results are presented in table 4.

 Example 5. The proposed method determines the activity of several series of purified and partially purified toxins of cholera vibrios. The control is the activity determined by the Craig method (see table 5).

 Thus, regardless of the purification of the drug, there is a clear coincidence of the results obtained by the proposed method and the standard method for animals.

 Example 6. The study of the activity of toxic shock exotoxin (ETS).

A mixture consisting of 0.8 ml of 2% NaCl and 0.1 ml of luminescent bacteria is added to a test tube containing 0.1 ml of ETS. The mixture is incubated at t 37 ^o C. The control is ETSh mixed with antitoxic serum (obtained by the method of Tekutiev, SI Karnitskaya NV. Study of ETS isolated from strain S. aureus FRI-1169. Sat. Bacterial toxins. Jurmala 1989 , v. 2, pp. 130-131). The results of the research are presented in table.6.

 The data table. 6 indicate that ETS inhibits the luminescence of bacteria.

 Example 7. The method described above determines the possibility of neutralizing ETS with specific antitoxic serum. The results of the determinations are presented in table.7. They indicate that specific serum neutralizes the effect of ETS. Thus, the proposed method allows to determine the activity of not only cholerogen, but also other toxins.

 The optimal exposure was 60 minutes. A longer exposure is impractical, and a shorter one does not give reliable results (see table 8).

 Example 8. In order to confirm the specificity of the reaction, experiments were carried out with endotoxins of Klebsiella and cholera vibrio, as well as other biologically active substances.

In a test tube containing 0.1 ml of a solution of the substance, make a mixture consisting of 0.8 ml of 2% NaCl and 0.1 ml of a solution of luminescent bacteria. The mixture was incubated for 60 min at t 37 ^o C. The results of the studies are presented in table.9.

 From table 9 it is seen that suppression of luminescence is a specific property of exotoxins, since other biologically active substances of protein and lipopolysaccharide nature do not incubate luminescence, and in some cases it stimulates.

 Thus, the proposed method is simple to implement, cheap and allows you to get an answer within 1 1.6 hours.

Claims (1)

 A method for determining the activity of bacterial toxins, including exposure to the studied toxin in different dilutions on a biological object, followed by evaluation of the result of the exposure, characterized in that the luminescent bacteria included in the Ekolyum kit (BioChemMac) are used as the object of exposure, and the studied toxin is incubated with luminescent bacteria for at least 30 minutes, an assessment of activity is carried out to the degree of inhibition of luminescence of the bacterium.

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